Biochemical Reconstitution of Ca2+-Dependent Exosome Secretion in Permeabilized Mammalian Cells

Exosomes are a subpopulation of the heterogenous pool of extracellular vesicles that are secreted to the extracellular space. Exosomes have been purported to play a role in intercellular communication and have demonstrated utility as biomarkers for a variety of diseases. Despite broad interest in exosome biology, the conditions that regulate their secretion are incompletely understood. The goal of this procedure is to biochemically reconstitute exosome secretion in Streptolysin O (SLO)-permeabilized mammalian cells. This protocol describes the reconstitution of lyophilized SLO, preparation of cytosol and SLO-permeabilized cells, assembly of the biochemical reconstitution reaction, and quantification of exosome secretion using a sensitive luminescence-based assay. This biochemical reconstitution reaction can be utilized to characterize the molecular mechanisms by which different gene products regulate exosome secretion. Key features This protocol establishes a functional in vitro system to reconstitute exosome secretion in permeabilized mammalian cells upon addition of cytosol, ATP, GTP, and calcium (Ca2+).

Exosomes are a subpopulation of the heterogenous pool of extracellular vesicles that are secreted to the extracellular space.Exosomes have been purported to play a role in intercellular communication and have demonstrated utility as biomarkers for a variety of diseases.Despite broad interest in exosome biology, the conditions that regulate their secretion are incompletely understood.The goal of this procedure is to biochemically reconstitute exosome secretion in Streptolysin O (SLO)-permeabilized mammalian cells.This protocol describes the reconstitution of lyophilized SLO, preparation of cytosol and SLO-permeabilized cells, assembly of the biochemical reconstitution reaction, and quantification of exosome secretion using a sensitive luminescence-based assay.This biochemical reconstitution reaction can be utilized to characterize the molecular mechanisms by which different gene products regulate exosome secretion.

Background
Extracellular vesicles (EVs) encompass a diverse pool of membrane-enclosed compartments released by cells to the extracellular space (Colombo et al., 2014).Exosomes are an EV subpopulation that is secreted upon fusion of multivesicular bodies (MVBs) at the cell surface and they have been suggested to play a role in intercellular communication in both physiological and disease states (Harding et al., 1983;Fong et al., 2015;Hsu et al., 2017).Of particular interest is the selective and likely tissue-specific protein and small RNA composition of exosomes, which offers the prospect of their use as biomarkers for disease progression (Shurtleff et al., 2017; Driedonks and Nolte-'t Hoen, 2019; Upton et al., 2021).Despite an accumulating interest in exosomes, the molecular mechanisms that regulate and execute their secretion are not well understood.A recent study inserted nanoluciferase (Nluc) into the endogenous locus of the exosome marker protein CD63 to allow simple quantification of exosome secretion (Hikita et al., 2018).We modified this assay by the addition of a membrane-impermeable Nluc inhibitor to allow a distinction between cellular debris and bona fide CD63-positive EVs (exosomes) (Walker et al., 2017;Williams et al., 2023).Using this assay, we demonstrated that MVBs participate in Ca 2+ -dependent plasma membrane repair.12. Aspirate the transport buffer, replace with 500 μL of cold high-salt transport buffer, and gently shake the plate at 4 °C for 10 min on a lateral shaker.13.Aspirate the high-salt transport buffer, replace with 500 μL of cold transport buffer, and gently shake the plate at 4 °C for 10 min on a lateral shaker.mM ATP, 40 mM creatine phosphate, 0.2 mg/mL creatine phosphokinase), 0.15 mM GTP, 4 mg/mL cytosol, and 2 mM CaCl2. 2. Aspirate the wash buffer from step C13 and replace with 200 μL of the reaction mix for each condition.3. Incubate the 24-well plate on ice for 5 min.4. Place the entire 24-well plate in a 30 °C water bath for 2 min to stimulate exosome secretion.5. Place the 24-well plate back on ice and immediately load 100 μL of each reaction supernatant into an AcroPrep 96-well 0.4 μm filter plate placed on top of a 96-well collection plate.6. Centrifuge the AcroPrep 96-well 0.4 μm filter plate at 1,500× g for 1 min at 4 °C in an Eppendorf 5810 R centrifuge using a A-4-62 swinging bucket rotor with a plate adaptor.7.While waiting on the centrifuge run, add 100 μL of cold transport buffer + 2% TX-100 (containing protease inhibitors) to each well of cells within the 24-well plate (for a final TX-100 concentration of 1%) to lyse the cells.8.The filtrate collected from step D6 is used to measure exosome secretion, and the lysate from step D7 is used to normalize exosome secretion between sample conditions.

Data analysis
1.The formula to calculate the exosome production index (EPI) is as follows: The EPI can then be normalized to the desired control condition.Example data are presented in Figure 2.

General notes and troubleshooting
1. We have observed variability in the activity of commercial SLO preparations.We recommend titrating each batch of SLO to identify the optimal concentration required to permeabilize a majority of cells as depicted in Figure 1.If needed, trypan blue exclusion can be utilized to confirm cell permeabilization.2. During optimization of this reconstitution assay, we observed a high level of background due to insufficient cytosol depletion.The identity and concentration of the salt utilized for the high-salt wash (step C12) may need to be optimized through empirical testing.We recommend conducting immunoblot analysis for a cytosolic marker (e.g., GAPDH) before and after the high-salt wash to ensure efficient cytosol depletion.

Figure 2 .
Figure 2. Biochemical reconstitution of Ca 2+ -dependent exosome secretion.(A-B).Exosome secretion from Streptolysin O (SLO)-permeabilized CD63-Nluc cells was assessed under different reaction conditions.The experimental conditions are indicated below each column and refer to the conditions described in Table 1 ("B" indicates baseline).Data plotted represent the means from three independent experiments, and error bars represent each standard deviation.Statistical significance was evaluated in GraphPad Prism using an ANOVA (*p < 0.05, ***p < 0.001, ****p < 0.0001, and ns = not significant) (Modified from Williams et al., 2023).

. PBS + 1 mM EGTA (10 mL)
Note: Make fresh and store at 4 °C until use.

Table 1 . Sample calculation for biochemical exosome secretion reconstitution reactions.
Each column represents a single condition for the reconstitution reaction.The total volume for each reaction mix is 250 μL for one reaction replicate and pipetting error.Each row represents the component to be added to each reaction, and the volumes indicated are in microliter.Each reaction mix can be scaled for the desired number of reaction replicates.

1 .
For the luminescence measures to quantify exosome secretion: a. Add 50 μL of the filtrate from step D6 to a microcentrifuge tube.b.Add 100 μL of Nluc substrate/inhibitor master mix to the filtrate.c.Vortex the sample briefly (~1 s) and measure luminescence in a Promega Glowmax 20/20 luminometer.Remove the sample tube from the luminometer and add 1.5 μL of 10% TX-100 (for a final TX-100 concentration of 0.1%) to solubilize membranes.